Immunotherapy is gaining importance for the treatment and prevention of various human diseases, including infectious diseases and cancers.
Regarding immunotherapy in cancers, with the recent FDA approval of the Sipuleucel-T vaccine for prostate cancer, the feasibility of active immunization for the treatment of established cancer has been demonstrated.
It is now established that the immune system can recognize and to some extent eliminate tumor cells through different cells subsets including CD8 cytotoxic T lymphocytes (CTLs). Modulating the immune system in order to track and specifically destroy the tumor cells is a promising therapeutic approach (also called anti-tumoral immunotherapy) for treating patients.
Tumor-associated antigens recognized by CTLs are 8 to 11 residue peptides called CD8+ epitopes which are bound to MHC class I molecules and displayed at the tumor cell surface. In the last decade, an increasing number of these peptides derived from the processing of tumor proteins have been identified and classified as tumor specific antigens (TSA) or tumor-associated antigens (TAAs). The main goal of current research on immunotherapy approaches is to elicit potent anti-tumor immunity after therapeutic vaccination against these antigens. Approaches widely developed and transferred to clinical trials include peptide vaccination and adoptive immunotherapy with ex-vivo loaded dendritic cells (DCs).
However, the clinical successes of these approaches have been modest. Among other reasons, this failure can be explained both by the very immunosuppressive properties of the tumor microenvironment and by the different immune escape mechanisms developed by the tumor cells including the loss of individual antigens.
Recently, the key role of another subset of T cells, called CD4+ helper T cells (Th), has been described in anti-tumor immunity. Indeed, it has been reported that this CD4 compartment plays a crucial role in mounting an efficient anti-tumoral immune response (Bos and Sherman, 2010, Cancer Res. 70:8368-8377). As for CD8 T cells, Th cells are also involved in the maintenance of long-lasting cellular immunity (immunological memory), and tumor infiltration by Th cells is an essential step for the recruitment and function of CTLs.
Tumor-associated antigens recognized by Th cells are typically 12-25 residue peptides (although some are much longer) called CD4+ epitopes which are bound to MHC class II molecules and displayed at the tumor cell surface.
The use of protein rather than peptides to induce anti-tumor immunity would allow multi-epitopic (CD8+ and CD4+ epitopes) antigen delivery to antigen presenting cells (APCs) such as dendritic cells (DCs). However, protein uptake by APCs is limited and frequently results in presentation of only CD4 epitopes by MHC class II molecules. This is because protein antigens taken up from the extracellular milieu do not efficiently enter the cytoplasm from where their constituent peptide epitopes can bind to MHC class I molecules being assembled in the endoplasmic reticulum (a process called cross-presentation). Therefore, there is a need to develop new approaches to increase the efficiency of protein uptake by DCs, and to facilitate presentation of both CD4 and CD8 epitopes.
Different vectors have been developed and evaluated to deliver different MHC class I restricted epitopes; these include viral vectors (Durantez et al., 2009, Scand. J. Immunol 69.80-89; Mateo et al., 1999, J. Immunol. 163:4058-4063; Tine et al., 2005, Vaccine 23:1085-1091), cDNA-based vaccine (Ishioka et al., 1999, J. Immunol. 162:3915-3925; Scardino et al, 2007, Cancer Res. 67:7028-7036) and mRNA electroporated dendritic cells (Waeckerle-Men et al., 2006, Cancer Immunol. Immunother. 55:1524-1533).
In addition to minimizing immune escape, targeting multiple epitopes allows a greater proportion of tumor cells in a heterogeneous tumor (i.e. different individual tumor cells expressing different antigens within same tumor) to be attacked. Some progress has been made for vaccinia virus vectors encoding multiple epitopes associated with infectious diseases (Thomson et al., 1996, J. Immunol. 157:822-826; Thomson et al., 1995, Proc. Natl. Acad. Sci. USA. 92:5845-5849; Anton et al., 1997, J. Immunol. 158:2535-2542). However, several limitations have been noted. The first is that vaccinia virus vectors encoded antigens are preferentially presented by MHC class I restricted molecules; second, there is a limitation of the size of insert; third, there is rapid degradation of the encoded antigens, and finally there are many regulatory hurdles for clinical translation.
An alternative approach that has several inherent advantages is a multi-epitope vaccine based on protein rather than on a viral or DNA based vaccine. This offers the major advantage of long-lasting MHC presentation of the cargo antigens to T lymphocytes (van Montfoort et al., 2009, Proc. Natl. Acad. Sci. USA. 106:6730-6735), but low immunogenicity of the vector—allowing for multiple vaccinations.
In the past decade, protein transduction domains (PTDs) are emerging as promising vectors to deliver different therapeutic targets, including proteins. PTDs are peptide sequences facilitating efficient protein translocation across biological membranes, independently of transporters or specific receptors. PTDs also offer the advantage of cost-efficient production. Since the discovery 20 years ago of the membrane translocating property of human immunodeficiency virus transactivating regulatory protein (HIV TAT), several PTDs have been identified including penetratin (Antennapedia homeodomain), VP22 (Herpes simplex virus) and the synthetic polyarginine (polyR). Different cargoes have been linked to PTD with the perspective of novel vaccine design. These include tumor-associated antigen for cancer immunotherapy.
The most widely studied PTD, TAT, was fused to different antigens and used to transduce dendritic cells (in virtually all studies) before testing immunogenicity in vivo (Brooks et al., 2010, Biochimica et Biophysica Acta 1805:25-34). In all these studies, a CTL-mediated immune response (i.e. mediated by CD8 T cells and restricted by MHC class I) was demonstrated after loading the DCs with the TAT-fusion protein, in contrast to the protein alone, and in some cases, CD4 T cells were also implicated. Moreover, vaccination with TAT fused to TRP2 resulted in long-term protection as shown in tumor-free mice re-challenged with the tumor, suggesting a superior memory response. However, there are several potential drawbacks concerning TAT. The first is that the use of TAT based vaccines directly in vivo without prior transduction of DCs remains largely unexplored. The second is that the nature of the cargo transported into the cell by TAT influences intracellular localisation; large TAT-fusion proteins can remain entrapped in endosomes where they are degraded, which is predicted to limit access to the cross-presentation pathway resulting in poor stimulation of CD8 T cells (Tünnemann et al., 2006, FASEB J., 20: 1775-1784).
Therefore, there is still a need for developing anti-tumor and anti-pathogen vaccines able to induce strong and broad T-cell responses specific for multiple epitopes of a given antigen, involving both CD4+ and CD8+ cells, preferably applicable to a broad range of patients, and that have the potential for direct injection into patients, without requiring DCs. The present invention solves this problem by providing a PTD fusion protein allowing efficient delivery and presentation of multiple CD4+- and CD8+-restricted epitopes. The multi-epitopic PTD fusion protein of the invention, thus, is useful in immunotherapy for treating and/or preventing cancers or infectious diseases.